The invention relates to the use of the combination of a lyase with a selenium-containing lyase substrate to inhibit or destroy tumor cells in vitro and in vivo. More specifically, the invention concerns providing an enzyme capable of generating a toxic selenium form from a relatively nontoxic substrate in combination with said substrate. An illustrative combination is selenomethionine in combination with methioninase. The enzyme may be supplied directly or generated recombinantly in situ.
Selenium is an element essential for metabolism, but in the wrong form and in inappropriate amounts it is cytotoxic. This may be advantageous in the context of carcinostatic properties. Spallholz, J. E., in a review article published in Free Radical Biology and Medicine (1994) 17:45-64, proposed that the toxic selenium species is the metabolic selenide RSexe2x88x92 anion. The anion can participate in transthiolation reactions and generate superoxide, hydrogen peroxide and perhaps other cascading oxyradicals, according to this proposal. In a later paper from the same group, Stewart, M. J., et al., Free Radical Biology and Medicine (1999) 26:42-48, it is reported that catalytic selenite, selenocystine and selenocystamine induced apoptosis and were cytotoxic in keratinocytes, but selenomethionine was not cytotoxic and did not induce cellular apoptosis at the concentrations studied. Selenomethionine has also been shown to act as a cancer chemoprotectant by Ip, et al., Cancer Res. (1990) 50:1206-1211; El-Bayoumy, K., et al., Cancer Res. (1992) 52:2402-2407. Selenium compounds shown to inhibit carcinogenesis in rodents have been reported by Yan, Y., et aL, Biochem. Pharmocol. (1993) 45:429-437.
Apparently, diselenide forms of organic selenium compounds, such as selenocystine and selenocystamine can be converted to the toxic form RSexe2x88x92. However, mammalian cells do not readily convert the monoselenide forms to toxic moieties. Thus, selenomethionine, selenohomocysteine and seleriocysteine are relatively nontoxic and noricarcinostatic.
Selenodithiols have been used to inhibit proliferation of cancer cells as described in U.S. Pat. No. 5,104,852 and selenium compounds which would otherwise be toxic have been used therapeutically in small enough concentrations as described in U.S. Pat. No. 4,512,977.
It has now been found that a particularly effective protocol for treatment of tumors comprises using the nontoxic forms of selenium as prodrugs when the targeted cells are provided with a means to convert the pro-drugs to the toxic forms. The pro-drugs themselves can be cytotoxic if at sufficiently high levels. Kajander, E. O., et al., Biochem. J (1990) 267:767-774. However, these levels are several orders of magnitude higher than those required for cytotoxicity of compounds that can readily generate RSexe2x88x92 in cells.
The invention is directed to protocols and formulations for selectively killing cancer cells through the combination of an RSexe2x88x92 generating enzyme, such as methioninase and its selenium-containing substrate, e.g., selenomethionine. The method can be used in chemotherapy regimen to treat a variety of cancers. The selenium-containing prodrug and/or the methioninase can also be selectively targeted to a particular cancer cell type or to a tumor site. The methioninase can be administered as a nucleic acid comprising a nucleotide sequence which encodes the enzyme that is expressible by the cancer cells or by other target host cells. The methioninase and the selenium-containing prodrug can also be directly administered to the tumor site, where possible or desired. The protocols cause cell death or retard cell growth.
The selenium-containing prodrugs include selenomethionine and related compounds. The prodrug is cleaved by a lyase at the C-Se bond to result in the release of. the active selenide moiety. This moiety is thought to be a methylselonide or a related compound of the formula RSexe2x88x92 where R is hydrocarbyl or H.
The enzyme administered will be one which serves as a lyase for the selenium-containing prodrug. If the selenium-containing prodrug is selenomethionine, methioninase is an appropriate choice.
Methioninase can be recombinantly produced or purified from natural sources. For example see U.S. Pat. No. 5,690,929. An expression system or cassette for production of methioninase can be used in place of the enzyme or along with it if desired. The vector permits the selective expression of the enzyme, which would localize its presence at a selected or desired site. Further, the methioninase can be complexed with a targeting molecule, e.g. an antibody that recognizes cancer cells. See, e.g. U.S. Pat. No. 5,057,313. The selenium-containing prodrug can similarly be targeted.
Thus, in one aspect, the invention is directed to a method to inhibit tumor cell growth which method comprises contacting said tumor cells with a selenium-containing prodrug and with an enzyme that liberates a toxic form of selenium from said prodrug in amounts sufficient to inhibit said tumor cell growth. In a preferred embodiment, the enzyme is an xcex1,xcex3-lyase or an xcex1,xcex2-lyase and the selenium-containing prodrug is a corresponding sulfur-containing amino acid or the corresponding amine. In one preferred embodiment, the lyase is supplied by generating it internally to the targeted tumor cells or in cells adjacent to them by providing a suitable: expression system to said cells or their neighbors.